ABSTRACT Apolipoprotein E (ApoE) genotype, the primary carrier of cholesterol within the brain, is an important determinant of an individual's risk for developing Alzheimer's disease (AD). While the ?2 allele (ApoE2) appears to be protective, ApoE4 increases the risk for the disease. Growing evidence, including our findings within this application, argue that ApoE genotype affects the endosomal and exosomal pathways. In young ApoE2 mice, our preliminary studies have shown that neuronal early endosomes are reduced in size and number while a greater number of exosomes are seen in the brain extracellular space as compared to mice expressing the human ApoE3 allele, the neutral-risk allele in humans, or mice expressing the pathogenic ApoE4 allele, which leads to abnormally enlarged early endosomes and lower levels of exosomes in older mice. We propose that exosomes, secreted vesicles generated within endosomal compartments, help clear endosomal vesicular content from the endocytic pathway, thus contributing to the preservation of neuronal endosomal function during aging. Additionally, we hypothesize that the facile movement of cargo through the endocytic pathway in ApoE2 carriers contributes to efficient lysosomal function. This combination of endosomal and exosomal changes driven by ApoE2 suggests interrelated, and potentially additive, neuroprotective cellular processes. We propose to test our hypothesis that expression of the ApoE2 allele results in protective changes within the endosomal and exosomal pathways by examining humanized ApoE mouse brain at various ages as well as ApoE genotyped human brain tissue (Aim 1). In brain-derived endosomes and exosomes we will examine the lipidome and proteome and in exosomes, RNAs to identify molecular alterations that mediate protective ApoE2 endosomal and exosomal functions. To tie ApoE2 cell biology to brain function we focus on the olfactory system. Several recent studies, including both mouse and human data in part from our group, demonstrate that olfactory sensory physiology and perception may be especially sensitive biomarkers of ApoE genotype. For example, ApoE4 carriers have impaired odor identification (humans), impaired odor memory and olfactory system hyperexcitability (mice). In Aim 2, we will examine both olfactory sensory physiology and behavior to test the hypothesis that the ApoE2 isoform modulates olfactory function and can rescue ApoE4 deficits. Finally, in preliminary studies we have shown that a high fat/cholesterol diet causes neuronal endosomal pathway pathology in wild-type mice. In Aim 3, we will test our hypothesis that ApoE2-induced efficiencies in the endosomal and exosomal pathways protect from dietary lipid-induced endosomal pathway abnormalities. The overall objectives of our study are to characterize interconnected cellular pathways that we propose are protective in individuals expressing ApoE2 and to determine whether maintaining efficient endosomal and exosomal function through ApoE2 expression reduces neuronal vulnerability and cognitive deficits during aging, focusing in part on the highly informative olfactory system.